The cystatin superfamily includes a number of cysteine protease inhibitors that are widely distributed in tissues and body fluids of mammalians (6). Chicken cystatin, a well-studied cysteine protease inhibitor, is a small non-glycosylated protein having 116 amino acids linked with two disulphide bonds (1, 5, 6, 18, 34), which has been crystallized and subjected to the preliminary X-ray crystallographic studies (10, 11). Chicken cystatin is a reverse, tight-binding inhibitor of cysteine proteases such as papain and tissue protease B and L, and is considered to contribute to physiological control in which said proteases participate (7, 8, 9, 23, 24, 26, 31). Chicken cystatin inhibits the degradation of proteins in organisms and the softening of muscles of animals when said animals were dead. In food industries, for example, chicken cystatin is added to surimi for the inhibition of endogenous proteolysis of the surimi, thereby reducing the gel softening of said surimi.
However, it was found that chicken cystatin is stable to heat (18), but unstable to freezing or freeze-drying (1, 20). Application of chicken cystatin on the inhibition of autolysis or endogenous proteolysis was greatly restricted by its lower, unsatisfactory freezing tolerance. Especially, chicken cystatin, when added to surimi, is unstable and easily loses its activity in the freezing-thawing process of surimi. For expanding the use of the chicken cystatin in frozen products, the structure of chicken cystatin may need to be modified for increasing its flexibility and resistance to a freezing-thawing process.
Natural chicken cystatin is not a glycoprotein since there is no N-glycosylation site in its amino acid sequence. There was no teaching or suggestion in the art that change in one or more amino acid residues in the amino acid sequence of chicken cystatin could improve the stability of chicken cystatin to temperature. Further, there was no teaching or suggestion in the art that glycosylation of the changed amino acid residue(s) in the amino acid sequence of chicken cystatin could improve the stability of chicken cystatin to temperature.
The inventors of the present invention found that change in just an internal amino acid residue of chicken cystatin that is not located in its active site, i.e. Asn106-Ile108→Asn106-Thr108, by employing site-directed mutagenesis and genetic engineering technique without changing or destroying the stereo structure of the active site of chicken cystatin, could facilitate the glycosylation of said Asn106, whereby the stability of chicken cystatin to temperature could be significantly improved. The Asn106-glycosylated, modified chicken cystatin of the present invention, when for example added to surimi, has the desired inhibitory function even if said surimi is treated with several freezing-thawing cycles.